See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/348590374

Nanopore-based metagenomics analysis reveals prevalence of mobile

antibiotic and heavy metal resistome in wastewater

Article  in  Ecotoxicology · October 2021

DOI: 10.1007/s10646-020-02342-w

CITATIONS READS

9 953

8 authors, including:

Steve Hamner Nur A Hasan

Montana State University EzBiome Inc
18 PUBLICATIONS   464 CITATIONS    189 PUBLICATIONS   5,860 CITATIONS   

SEE PROFILE SEE PROFILE

Rita R Colwell Timothy E Ford

University of Maryland, College Park University of Massachusetts Lowell
1,105 PUBLICATIONS   70,408 CITATIONS    207 PUBLICATIONS   6,275 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Psychrophilic/psychrotolerant bacteria View project

Biodeterioration of polymeric materials View project

All content following this page was uploaded by Nur A Hasan on 01 February 2022.

The user has requested enhancement of the downloaded file.

 Ecotoxicology (2021) 30:1572–1585
https://doi.org/10.1007/s10646-020-02342-w

Nanopore-based metagenomics analysis reveals prevalence of

mobile antibiotic and heavy metal resistome in wastewater
Cristina Martin1 Brooke Stebbins2 Asha Ajmani3 Arianna Comendul4 Steve Hamner5 Nur A. Hasan6
● ● ● ● ● ●

Rita Colwell6 Timothy Ford7

●

Accepted: 25 December 2020 / Published online: 18 January 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
In-depth studies of the microbiome and mobile resistome profile of different environments is central to understanding the
role of the environment in antimicrobial resistance (AMR), which is one of the urgent threats to global public health. In this
study, we demonstrated the use of a rapid (and easily portable) sequencing approach coupled with user-friendly
bioinformatics tools, the MinION (Oxford Nanopore Technologies), on the evaluation of the microbial as well as mobile
metal and antibiotic resistome profile of semi-rural wastewater. A total of 20 unique phyla, 43 classes, 227 genera, and
1234567890();,:
1234567890();,:

469 species were identified in samples collected from the Amherst Wastewater Treatment Plant, both from primary and
secondary treated wastewater. Alpha diversity indices indicated that primary samples were significantly richer and more
microbially diverse than secondary samples. A total of 1041 ARGs, 68 MRGs, and 17 MGEs were detected in this study.
There were more classes of AMR genes in primary than secondary wastewater, but in both cases multidrug, beta-lactam and
peptide AMR predominated. Of note, OXA β-lactamases, some of which are also carbapenemases, were enriched in
secondary samples. Metal resistance genes against arsenic, copper, zinc and molybdenum were the dominant MRGs in the
majority of the samples. A larger proportion of resistome genes were located in chromosome-derived sequences except for
mobilome genes, which were predominantly located in plasmid-derived sequences. Genetic elements related to transposase
were the most common MGEs in all samples. Mobile or MGE/plasmid-associated resistome genes that confer resistance to
last resort antimicrobials such as carbapenems and colistin were detected in most samples. Worryingly, several of these
potentially transferable genes were found to be carried by clinically-relevant hosts including pathogenic bacterial species in
the orders Aeromonadales, Clostridiales, Enterobacterales and Pseudomonadales. This study demonstrated that the MinION
can be used as a metagenomics approach to evaluate the microbiome, resistome, and mobilome profile of primary and
secondary wastewater.
Keywords Resistome Mobilome Wastewater Metagenome MinION
● ● ● ●

Introduction

The overuse and misuse of antimicrobials has accelerated

the naturally existing phenomenon of antimicrobial resis-
* Timothy Ford
timothy_ford@uml.edu
tance (AMR) and resulted in new mechanisms that can
bypass the current arsenal of “last-resort” antimicrobials,
1
Manila, Philippines including colistin (D’Costa et al. 2011; Thi Khanh Nhu
2
Portland Water Bureau, Portland, OR 97227, USA et al. 2016; Mataseje et al. 2014; Friedrich 2016). The UN
3 Ad hoc Interagency Coordinating Group (IACG) on Anti-
University of Massachusetts Amherst, Amherst, MA 01003, USA
4
microbial Resistance estimates that drug-resistant diseases
Partners In Health, Boston, MA 02199, USA
will cause roughly 10 million deaths per annum by 2050
5
Gonzaga University, Spokane, WA 99258, USA (IACG 2018; O’Neill 2014). Thus, the WHO now considers
6
Center for Bioinformatics and Computational Biology, University AMR an urgent global public health concern (WHO 2014).
of Maryland, College Park, MD 20742, USA Active AMR surveillance, including environmental sur-
7
University of Massachusetts Lowell, Lowell, MA 01854, USA veillance, is critical for protecting public health (WHO
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . .
2014). A rapid, cost-efficient, but sufficiently comprehen-
sive method of environmental AMR surveillance will make
regular monitoring feasible and will allow for in-depth
studies of different environments, which is central to
understanding the role of the environment in AMR.
AMR and the resistome are broad terms that include
antibiotic resistance genes among several others. In this
study, the resistome will only refer to antibiotic resistance
and metal resistance genes while the mobile genetic ele-
ments like plasmids, transposons, and integrons are referred
to as the mobilome. As the potential health risk posed by
environmental AMR genes is related to their mobility
(Stokes and Gillings 2011; Ashbolt et al. 2013; Leonard
et al. 2018; Cantón et al. 2012; von Wintersdorff et al.
2016), an effective surveillance strategy would need to
include not only microbial resistome and resistome host
characterization, but assessment of the mobile resistome as
well. Several studies have employed a similar strategy,
using culture-based methods together with metagenomics
sequencing, employing both short and long-read technolo-
gies (Zhao et al. 2015; Xia et al. 2017; Luo et al. 2017; Che
et al. 2019), or PCR-based methods (Hultman et al. 2018).
Long-read sequencing technology such as those offered
by Oxford Nanopore Technology (ONT) have the dis-
advantage of base inaccuracy due to relatively high
sequencing error rates (Jain et al. 2016) compared to short-
read sequencing, but offer the advantage of structural
accuracy due to the generation of assembly-free long con-
tigs (Urban et al. 2015). The ONT platform also allows for
generation of data in real time, making rapid surveillance
methods possible. In addition, the ONT MinION technol-
ogy is highly portable, avoiding the necessity of access to
major sequencing facilities (Mongan et al. 2020). Coupling
ONT technologies with a bioinformatics pipeline like
NanoARG (Arango-Argoty et al. 2019), which is user-
friendly and comprehensive enough to provide information
on the (1) microbiome, (2) resistome, and (3) mobilome,
creates a rapid and sufficiently thorough surveillance
method.
The wastewater system is an ideal environment to initiate
AMR surveillance as it brings together the pathogenic and
non-pathogenic microbes, resistance and mobile genetic
elements, antimicrobials, and other chemicals that might
further contribute to mobile AMR selection (Pehrsson et al.
2016). Moreover, wastewater effluents have been shown to
contribute to the spread of AMR genes (Lamba and
Ahammad 2017; Berendonk et al. 2015; An et al. 2018;
LaPara et al. 2011; Czekalski et al. 2012), making it even
more relevant to public health. Here, we demonstrate the
use of a rapid, sequencing approach for monitoring and
evaluation of the microbial community, as well as the
mobile metal and antibiotic resistome profile of semi-rural
Amherst wastewater in western Massachusetts. We also
1573
investigated the potential effects of the wastewater treat-
ment process on the above-mentioned profiles.
Materials and methods
Wastewater sampling
Primary and secondary wastewater effluent were collected
in triplicate in March and April 2019 using sterile 250-mL
polypropylene bottles (Nalgene™). The triplicate samples
were collected the same day within a one hour timeframe.
The samples were either immediately processed or stored at
−20 °C.
Wastewater sample processing: filtration, DNA
extraction and library preparation
The samples were mixed thoroughly and 100 mL filtered
using sterile 0.22 micron MCE filter membranes (Mem-
brane Solutions Corp) and a Pall vacuum manifold (Pall
Corporation). The filters were loosely rolled and placed
inside bead tubes and DNA extracted following the Pow-
erWater DNA Isolation Kit protocol (Qiagen). The quantity
and quality of the isolated DNA were determined using a
NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher
Scientific). The initial amount of genomic DNA per sample
was 400–500 ng. DNA libraries for multiplex sequencing
were prepared without shearing using the Rapid Barcoding
Kit SQK-RBK004 (ONT, Oxford, UK) following the sup-
plied protocol. Sample clean-up was performed with
AMPure beads (New England Biolabs, MA, USA) prior to
addition of the adapter mix (RAP) included in the barcoding
kit. The purity (A260/A280 ≥ 1.8) and quantity (≥400 ng) of
the DNA samples in this study met the minimum require-
ments for MinION sequencing (ONT), with the exception of
the second set of effluent samples (April secondary
samples).
Shotgun metagenomic sequencing and
bioinformatics analysis
The pooled library consisting of 12 barcoded samples was
loaded onto a flow cell with R9.4.1 pore chemistry (Oxford
Nanopore FLO-MIN106). The flow cell had pore counts
greater than 1500 prior to sequencing. Shotgun metage-
nomic DNA sequencing was performed for 48 h with the
Oxford Nanopore MinION platform using the NC_48
h_Sequencing_Run_FLO-MIN106_SQK-RBK004 protocol
and raw output signals were base called in real time using
the MinKnow software (ONT; version 18.12). The 48-h run
generated a total of 1,492,345 1D reads, with an average
sequence length of 2271 basepairs (bp), a maximum length
1574
of 22,587 bp and a total yield of 3.4 Gigabases. The fastq
files were delivered to CosmosID (www.cosmosid.com;
MD, USA) for data processing and bioinformatics analysis
to identify the microbial diversity, virulence factors, and
antimicrobial resistance genes. For comparison, the same
files were uploaded into Epi2Me for cloud-based processing
and bioinformatics analysis (ONT’s Metrichor). The cloud-
based WIMP (What’s In My Pot) and ARMA (Anti-
microbial Resistance Mapping Application) workflows in
the Epi2Me program (ONT) were used for taxonomic
identification and characterization of antimicrobial resis-
tance genes respectively. The WIMP workflow aligns
MinION reads using minimap2 (Li 2018) against the Cen-
trifuge database (Kim et al. 2016) and ARMA aligns reads
using LAST (Kiełbasa et al. 2011) against the Compre-
hensive Antibiotic Resistance Database (CARD; McArthur
et al. 2013).
In order to perform further bioinformatic analyses to
identify the resistome and mobilome in plasmid-derived
versus chromosome-derived sequence reads, the raw multi-
read fast5 files were first converted to single read files using
the Python script ont_fast5_api (ONT; version 1.4.4) and
then basecalled with the command-line basecaller Guppy
(ONT; version 3.2.2) using the default parameters for the
FLO-MIN106 flow cell/SQK-RBK004 kit. The base called
reads were demultiplexed using the realtime default para-
meters of DeepBinner (version 0.2.0; Wick et al. 2018) for
the SQK-RBK004 kit. NanoPlot (version 1.28.2; De Coster
et al. 2018) was used to check the sequence read quality.
Filtlong (version 0.2.0; https://github.com/rrwick/Filtlong)
was used to filter out reads less than 1 kilobase (kb) in
length and to remove the lowest 10% of the reads based on
the Phred quality scores. The barcodes and adapters were
then removed using the default parameters of PoreChop
(version 0.2.3; https://github.com/rrwick/Porechop). These
filtered and trimmed reads were then uploaded into the
Galaxy server (Afgan et al. 2016), converted to FASTA
format, and analyzed to sort out plasmid- and chromosome-
derived read sequences using the default settings of Plas-
Flow (version 1.0; Krawczyk et al. 2018). PlasFlow analysis
produced three sets of data: chromosome-derived, plasmid-
derived, and unclassified. All three outputs were submitted
for further analysis through the NanoARG (Arango-Argoty
et al. 2019) workflow to identify the mobile genetic ele-
ments (MGEs), antimicrobial resistance genes, and metal
resistance genes.
Briefly, the NanoARG workflow initially clusters the
sequence reads using the permissive parameters of DIA-
MOND (Buchfink et al. 2015). To annotate antibiotic
resistance genes (ARGs), NanoARG employs the
DeepARG-LS (Arango-Argoty et al. 2018) method to query
the more extensive and consolidated DeepARG-DB data-
base. To annotate metal resistance genes (MRGs),
C. Martin et al.
NanoARG uses the BacMet database (Pal et al. 2014), and
for MGEs it uses the MGE database from the NCBI-NR and
integron-integrase (I-VIP; Zhang et al. 2018) databases.
NanoARG performs taxonomic identification using the
default parameters of Centrifuge (Kim et al. 2016) and then
against the NCBI-NR and ESKAPE/WHO database for
identification of potential critical pathogens.
Statistical analysis
All data processing, statistical analyses and plotting were
done in R (version 4.0.2) using the following R packages:
vegan (v2.5-6), phyloseq (v1.32.0), mixOmics (v6.12.2),
ggplot2 (v3.3.2), zCompositions (v1.3.4), compositions
(v2.0.0), and ALDEx2 (v1.20.0).
Metagenomic data
All metagenomic data (FastQ Files) have been deposited in
the NCBI SRA database with BioProject ID:
PRJNA684899 and accession number SAMN17054264.
Results
Microbial community composition
Multiplexed, whole-genome shotgun metagenomic
sequencing using ONT’s MinION platform was used to
study the microbial community of Amherst wastewater
samples. CosmosID identified a total of five unique king-
doms, 20 phyla, 43 classes, 227 genera, and 469 species
across all wastewater samples. Bacteria comprised 93% of
the kingdom followed by viruses (5%) and Archaea (0.5%).
Of the Archaea, the genus Methanobrevibacter was mostly
found in primary samples and the genus Methanosarcina
was found only in secondary samples. A similar pattern was
also observed for Fungi (1%) and Eukaryota (0.7%), in that
the majority were detected in primary samples. About 75%
of the bacteria were gram-negative across all samples,
consistent with results from ONT’s WIMP analysis (Fig. 1).
A number of Operational Taxonomic Units (OTUs; about
45% of the bacteria detected and identified in all samples)
represented human and animal pathogens, a majority of
which were gram-negative (Fig. 1).
Proteobacteria was the dominant phylum (~44%) in all
samples followed by Bacteroidetes (25–28%), although
there was variation with respect to relative abundance and
less diversity within secondary effluent samples at the class
level (Fig. 2a, b). Both Fig. 2a and b showed a distinct
pattern for primary samples compared to the secondary
samples. Microbial species belonging to the phylum Acti-
nobacteria were more prominent in secondary samples than
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . .
Fig. 1 Divergent barplot created
using relative abundances shows
distribution of Gram-negative
and Gram-positive pathogens
and non-pathogens in primary
(blue) and secondary (orange)
wastewater
primary samples (~12% compared to 2%, respectively). The
opposite was observed for Cyanobacteria, i.e., 1.4% in
primary samples but non-detectable in secondary samples.
Viruses were detected in all primary samples but not in all
secondary samples. Four classes of Proteobacteria were
detected in the majority of the primary samples whereas
only Betaproteobacteria and Gammaproteobacteria were
observed in secondary samples (Fig. 2b). A similar pattern
was obtained for Bacteroidetes, with three classes detected
and identified in primary effluent but only one, Bacteroidia,
in secondary effluent.
To assess microbial community structure, the raw microbial
read counts and the corresponding taxonomic information
obtained from CosmosID were consolidated and initially
explored using traditional diversity analysis methods and ana-
lyzed using the compositional data (CoDa) analysis approach.
Traditional approach
The untransformed microbial counts at the genus level were
initially explored to determine whether the overall data
showed separation related to wastewater treatment. The
alpha diversity metrics Chao1, Shannon and inverse
Simpson indices were used to assess richness and evenness
within and between primary and secondary wastewater
groups (Fig. 3a). Differences between sample groups for all
metrics were significant (Mann–Whitney U test, p < 0.01,
adjusted using the Bonferroni-Holm method), which indi-
cated that Amherst primary wastewater samples were more
diverse and microbially richer than the secondary samples.
A similar observation was also seen for data filtered using
1575
the phyloseq package in R, which removed roughly 55% of
the original data. The cluster analysis and principal coor-
dinates analysis (Fig. 3b, c) obtained from the Bray–Curtis
dissimilarity values indicated significant separation in
microbial taxonomic composition between primary and
secondary wastewater. A PERMANOVA analysis showed
44% of the variation between samples can be accounted for
by wastewater treatment. Beta diversity was 0.24
(SD = 0.09) within primary wastewater samples and 0.65
(SD = 0.30) within secondary samples, which increased to
0.76 (SD = 0.20) between primary and secondary samples
(PERMANOVA, p < 0.01).
Compositional data analysis
To account for compositionality of the sequencing data
(Gloor et al. 2017), clr (centered log-ratio) transformation
using the mixOmics package in R was applied in compo-
sitional data (CoDa) exploration and analysis. The zero
values in the raw counts data were imputed prior to trans-
formation using the count zero multiplicative (CZM)
method in the zCompositions package. The compositional
biplot shown in Fig. 4a and the unsupervised cluster ana-
lysis (generated with the ward.D2 method) clearly present
qualitative microbial structure separation at the genus level
between primary and secondary samples (Fig. 4b). Both the
primary and secondary samples are mainly clustered toge-
ther. The AS1 sample appeared to be an outlier in the biplot
but was not treated as such in this study because all other
analyses involving it were generally consistent with the rest
of the secondary samples (Table 1).
1576
Fig. 2 a, b Microbial composition of wastewater samples. Stacked
barplots show community composition of primary and secondary
wastewater at the a phylum level and b class level. The taxa were
arranged such that the order of the phyla in a corresponds to those of
its constituent microbial classes in b
Effect size was estimated using the ALDEx2 package
(Fernandes et al. 2014) to determine specific taxa (genus
level) that were statistically responsible for the observed
discrimination between wastewater treatment samples. Clr
transformation of the raw count data was also done using
the same package. The calculated parameters for significant
taxa are summarized in Table 2. The diff.btw values
represent median difference in clr values between primary
and secondary samples while diff.win values represent the
median of the largest difference in clr values within primary
and secondary samples. The overlap metric represents the
proportion of the effect size metric that overlaps zero; i.e., if
it overlaps zero, there’s no effect. Figure 5a shows the
distribution of taxa significantly different than the sample
mean after the Benjamini-Hochberg correction, which
controls for false positive identification. Taxa with higher
abundance values than the mean in secondary samples had
positive diff.btw values, while taxa with higher abundance
values than the mean in primary samples had negative diff.
C. Martin et al.
btw values. A total of 16 genera were significantly different
(adjusted p value < 0.05) between wastewater treatment
types (Fig. 5b). Nine out of 16 are associated with sec-
ondary samples, two of which, Bacteroides and Acineto-
bacter, are opportunistic pathogens.
Effect of wastewater treatment on resistome,
mobilome and mobile resistome
Prior to resistome and mobilome profiling using the
NanoARG pipeline, the metagenomic DNA sequence reads
were sorted into plasmid- and chromosome-derived
sequences (Table 3). A total of 1041 ARGs, 68 MRGs,
and 17 MGEs were detected across all samples in this study.
Both chromosome and plasmid-derived reads of secondary
samples contained higher relative abundance (computed
using the method of Ma et al. 2016) of total ARGs than their
counterparts in primary samples (Fig. 6a). Despite this, the
ARGs in secondary samples are significantly less diverse
(Fig. 6a; Table 4) (Mann–Whitney U test, p < 0.05, adjusted
using the Bonferroni–Holm method) both in terms of
unique ARG genes and unique ARG types (grouped
according to the antibiotic class a gene confers resistance
to). There are no significant differences in ARG richness
across samples. The ARG types identified included ami-
noglycosides, antimicrobial lipids, beta-lactams, diamino-
pyrimidines, fosfomycins, MLS, multidrug, peptide
antibiotics, phenicols, quinolones, sulfonamides and tetra-
cyclines. Multidrug (~34%), beta-lactam (~20%), and pep-
tide (~11%) ARGs predominated in both primary and
secondary wastewater samples.
The occurrence of ARGs for aminoglycosides, beta-lac-
tams, MLS, multidrug, peptide antibiotics, phenicols, and
quinolones mainly found in chromosome reads are sig-
nificantly higher for primary samples (Mann–Whitney U
test, p < 0.05, adjusted using the Bonferroni–Holm method).
The most frequent ARG genes in chromosome-derived
reads are those for multidrug and peptide antibiotics while
in plasmid-derived reads, resistance genes for multidrug and
beta-lactams have the highest occurrence. The NanoARG
pipeline demonstrated some consistency with the Cosmo-
sID workflow, which also identified aminoglycosides, beta-
lactams, MLS, multidrug (msrE), quinolones, and tetra-
cyclines. The Epi2Me workflow was less consistent, but
also identified aminoglycosides, multidrug (msrE etc), beta-
lactams, quinolones, tetracyclines and a variety of bacteria-
specific mutations for elfamycin resistance. Of note was the
finding that secondary samples for both March and April
were enriched in certain OXA β-lactamases.
As shown in Fig. 6a (PS versus PP), the occurrence of
beta-lactam resistance genes is noticeably higher in the
plasmid-derived reads of secondary samples. ARGs for
beta-lactams found in chromosome reads including carO,
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . . 1577

Fig. 3 a Alpha diversity indices

computed using the Phyloseq
(v1.32.0) package in R (v4.0.2)/
(Rstudio v1.3.959) indicate that
primary wastewater was
significantly richer and more
diverse than secondary
wastewater (Mann–Whitney
U test; p values adjusted using
Bonferroni–Holm method).
b Cluster analysis (ward.D2
method) and heatmap generated
in R using relative abundances
shows discrimination between
primary and secondary
wastewater treatment samples
with respect to microbial
composition at the Genus level.
c Principal coordinates analysis
was performed using the
Bray–Curtis metric in the Vegan
(v2.5-6) package to show the
significant dissimilarities in
microbial composition across
samples (PERMANOVA,
p < 0.01)

class A/C β-lactamases, Nmcr, OXA-12, OXA-198, and
PBP-1A/2X occur more significantly in primary samples,
whereas those found in plasmid reads like class A
β-lactamases, OXA-10, OXA-11,OXA-233 and OXA-246
are significantly higher in secondary samples
(Mann–Whitney U test, p < 0.05, adjusted using the
Bonferroni–Holm method). In both primary and secondary
samples, the NanoARG pipeline found more ARGs in
chromosome- than plasmid-derived reads (43–54% versus
16–24%; the rest are found in unclassified reads). Resis-
tance genes to antimicrobial lipids, diaminopyrimidines,
fosfomycins, and phenicols were mostly absent in the
plasmid-derived reads of all samples.
The alpha and beta diversity indices indicated no sig-
nificant difference between primary and secondary waste-
water in terms of overall MRG richness, diversity and
composition (Table 4). As observed for ARGs, more
MRGs are found in chromosome than plasmid-derived
reads (44–52% versus 18–19%; the rest are found in
unclassified reads)(Fig. 6b). The occurrence of MRGs for
aluminium, cobalt, cadmium, gold, lead, magnesium,
mercury, silver, tellurium, and tungsten are significantly
greater in primary samples (Mann–Whitney U test, p <
0.05, adjusted using the Bonferroni–Holm method). Metal
resistance genes (Fig. 6b) against arsenic, copper, zinc and
molybdenum were the dominant MRGs in the majority of
the samples.
For the overall resistome (ARGs and MRGs), primary
samples were significantly more diverse according to the
Shannon metric (Mann–Whitney U test, p = 0.04, adjusted
using the Bonferroni–Holm method) (Table 4). The resis-
tome composition was significantly different between pri-
mary and secondary samples (PERMANOVA, p = 0.039).
A larger proportion of resistome genes were located in
chromosome-derived sequences (Fig. 6a, b) except for
mobilome genes (Fig. 6c), which were predominantly
located in plasmid-derived sequences. The mobilome
diversity and composition was significantly higher in sec-
ondary samples (Table 4). However, the class 1 integron-
integrase gene intl1, which is the least frequent MGE in this
study, is exclusively found in the plasmid reads of primary
samples. Genetic elements related to transposase were the
most prominent MGE in all samples.
About 65% of the total resistome (58% of ARGs and
97% of MRGs) in this study are mobile resistome, which
are either found in plasmid reads, found with MGEs in the
same read or both. The most frequent mobile resistome
genes are resistance genes for beta-lactams, multidrug,
peptide antibiotics, molybdenum, arsenic and copper.
Mobile MRGs including resistance genes for cadmium,
1578 C. Martin et al.

Fig. 4 a, b Compositional biplot

and unsupervised cluster
dendrogram (ward.D2 method)
generated in R show qualitative
microbial structure separation at
the genus level between primary
and secondary samples. The
scree plot in a was used to
determine the number of factors
for principal component analysis

cobalt, copper, gold, iron, lead, manganese, mercury,
molybdenum, selenium, tellurium and zinc, and mobile
ARGs for aminoglycosides, beta-lactams, MLS, multidrug
and peptide antibiotics predominantly occur in primary
samples (Mann–Whitney U test, p < 0.05, adjusted using the
Bonferroni–Holm method). The overall mobile resistome
diversity and composition is significantly higher for primary
samples (Table 4). The NanoARG pipeline was able to
identify a few clinically-relevant mobile resistome-
associated bacterial hosts in all samples using the NCBI/
ESKAPE-WHO database. These include pathogenic species
in the bacterial orders Clostridiales, Enterobacterales,
Vibrionales, Aeromonadales, and Pseudomonadales. Sev-
eral of the mobile resistome genes carried in these hosts
include mcr and OXA variants, which confer resistance to
last resort antimicrobials like colistin and carbapenems.
Discussion
Effects of wastewater treatment on microbial
community composition
The wastewater system is important to public health as it
can serve as a major sink and source of pathogens con-
taining mobile AMR, as well as an indication of community
public health (Fouz et al. 2020; Kraemer et al. 2019). The
issue of AMR is regarded as a top global public health
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . . 1579

Table 1 Description of
Samples Sample labels Total readsa Treatment Barcodeb
wastewater samples and total
nucleic acid reads April Amherst WW influent AP1 116,950 Primary BC07
April Amherst WW influent AP2 66,033 Primary BC08
April Amherst WW influent AP3 106,023 Primary BC09
March Amherst WW influent MP1 120,115 Primary BC01
March Amherst WW influent MP2 95,987 Primary BC02
March Amherst WW influent MP3 121,196 Primary BC03
April Amherst WW effluent AS1 18,266 Secondary BC10
April Amherst WW effluent AS2 23,208 Secondary BC11
April Amherst WW effluent AS3 31,519 Secondary BC12
March Amherst WW effluent MS1 139,910 Secondary BC04
March Amherst WW effluent MS2 195,473 Secondary BC05
March Amherst WW effluent MS3 157,577 Secondary BC06
WW wastewater
a
As per CosmosID report summary
b
SQK-RBK004 kit (ONT)

Table 2 Parameters calculated after ALDEx2 size estimation of

significant taxa at the genus level that are statistically responsible for
the observed discrimination between wastewater treatments
Genus diff.btw diff.win Effect Overlap

Acinetobacter 2.803 1.091 2.614 0.000

Prevotella 3.099 1.298 2.308 0.010
Tetrasphaera 7.364 1.782 4.330 0.000
Azonexaceae 11.135 3.429 3.255 0.000
Polaromonas 7.381 2.751 2.990 0.000
Bacteroides 2.995 1.244 2.268 0.005
Ruminococcus 2.428 0.653 3.212 0.003
Flavobacterium −6.064 2.218 −2.750 0.000
Oscillatoria −6.734 3.295 −1.989 0.003
Yersinia −6.455 3.594 −1.636 0.010
Empedobacter −5.734 3.473 −1.413 0.031
Sebaldella −5.757 3.601 −1.387 0.034
Paenibacillus −7.217 3.419 −1.937 0.003
Myoviridae −5.775 3.349 −1.540 0.029
Nitrospira 11.662 5.903 1.859 0.036
Sediminibacterium 10.051 4.369 2.128 0.026
diff.btw median difference in clr values between primary and
secondary samples, diff.win median of the largest difference in clr
values within primary and secondary samples, effect median effect
size, overlap proportion of effect size that overlaps 0, i.e. no effect
Fig. 5 a ALDEx2 plot generated using the ALDEx2 (1.20.0) package
in R show distribution of taxa significantly different (red dots) from the
sample mean after the Benjamini–Hochberg correction for false
concern (IACG 2018; O’Neill 2014; WHO 2014) with positive rate. Taxa with higher abundance than the mean in secondary
AMR surveillance viewed as an essential step towards samples have positive diff.btw values, while taxa with higher abun-
addressing this issue (WHO 2014). This makes regular dance than the mean in primary wastewater yielded negative values.
b Divergent barplot show the 16 genera that were significantly
monitoring of wastewater systems more relevant than ever, different (Mann–Whitney U test, adjusted p value < 0.05) between
as it not only ensures the safety of effluents relevant to wastewater treatments
animal and human contact, but also allows for studies
investigating AMR patterns and potential transmission
mechanisms in human-impacted environments. Such
1580 C. Martin et al.

Table 3 Summary of
Sample labels Reads (NanoARG)
metagenomic DNA sequence
reads sorted into plasmid- and Total reads Chromosome Plasmid Unclassified
chromosome-derived sequences
using PlasFlow (version 1.0) AP1 51,048 20,399 (40%) 11,290 (11%) 19,359
AP2 26,824 10,673 (40%) 6195 (23%) 9956
AP3 49,782 19,260 (39%) 11,570 (23%) 18,952
MP1 53,549 22,483 (42%) 10,894 (20%) 20,172
MP2 47,999 20,037 (42%) 9701 (20%) 18,261
MP3 52,438 21,937 (42%) 10,508 (20%) 19,993
AS1 12,003 3718 (31%) 3301 (28%) 4984
AS2 10,252 3556 (35%) 2665 (26%) 4031
AS3 18,015 5965 (33%) 4707 (26%) 7343
MS1 61,850 19,730 (32%) 16,190 (26%) 25,930
MS2 95,756 30,224 (32%) 26,000 (27%) 39,532
MS3 72,364 24,237 (33%) 18,526 (26%) 29,601

studies, especially if done over long timeframes, can help
define the extent of the AMR problem, which is relevant in
resolving this global issue. In this study, we demonstrate a
rapid but sufficiently comprehensive metagenomic method
that can make long-term routine wastewater monitoring
more feasible.
Initial characterization of the wastewater samples in
terms of microbial diversity and composition revealed
substantial discrimination between primary and secondary
samples, which is expected as each sample underwent a
different treatment process. Primary treatment primarily
involves sedimentation-based removal whereas secondary
treatment involves filtration and biological-based processes
(Quach-Cu et al. 2018). Acinetobacter was found to be the
dominant genus in all samples, present in primary samples
at a slightly higher abundance of 23% compared to 14% in
secondary samples. The second most abundant genera dif-
fered by treatment sample—Bacteroides for primary sam-
ples and Polaromonas for secondary samples. Aeromonas
and Ruminococcus species were more prominently detected
in secondary samples whereas Arcobacter and Onygenales
were more abundant in primary samples. Bacteroides and
Ruminococcus are typical gut bacteria and Acinetobacter,
Polaromonas, Onygenales, Aeromonas, and Arcobacter are
environmental microbes. Several Aeromonas, Arcobacter,
and Acinetobacter species are known zoonotic pathogens.
Thus, the microbial composition of the wastewater samples
comprise a complex mixture of gut and environmental
microbes, a conclusion from other wastewater systems as
well (Newton et al. 2015; Numberger et al. 2019).
Microbial richness and diversity significantly decreased
with the wastewater treatment process, not unexpected
given that secondary effluent samples have passed through
wastewater treatment steps. However, the observed
decrease in microbial richness and diversity in secondary
samples did not translate to a relative reduction in
pathogenic species (Fig. 1). Although culture-based meth-
ods would determine actual viability, the data from this and
other studies (Chahal et al. 2016; Osińska et al. 2017;
Harnisz and Korzeniewska 2018) show that most waste-
water treatment processes are insufficient to remove all
potential pathogens from effluents.
Compositional data approach
The compositional data approach supported the initial obser-
vations from traditional data analyses, showing microbial
structure differences between primary and secondary waste-
water. The difference is that the CoDa analysis was mainly
done at the genus level for greater resolution of taxa involved
in the observed separation. Primary wastewater samples pre-
dominantly grouped together, suggesting they contained similar
taxa at similar abundances (Gloor and Reid 2016). Further
interpretation of the biplot is qualitative at best because only
78% of the variability is explained by the first two principal
components. Considering the limited data projection, a few
rules can be employed to interpret the biplot data. First, the
length of the ray is related to relative taxon variability (Gloor
and Reid 2016). The longer the ray, the more variable the
represented taxon relative to other taxa. Using this rule, it can
be seen that Acidovorax (Betaproteobacteria), Arcobacter
(Epsilonproteobacteria), Bacteroides (Bacteroidia), and Rumi-
nococcus (Clostridia) reveal relatively longer rays, hence
greater variability with respect to wastewater treatment than
other taxa in the biplot. Acinetobacter (Gammaproteobacteria)
had the shortest ray, thus the least variability. A second rule
concerns the angle between rays, namely correlation between
taxa. A smaller angle indicates correlation of abundance of the
involved taxa. Rays of taxa that are orthogonal are uncorre-
lated. Figure 4a shows a small angle between rays for
Acinetobacter-Prevotella and Acinetobacter-Bacteroides, sug-
gesting abundance of these taxa were highly correlated while
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . . 1581

Table 4 Summary of the p values calculated to determine statistical

difference of alpha and beta diversity indices for the different
groupings of resistomes and mobilomes along wastewater treatment
Alpha diversity (adj. p values)a Beta diversity
(p values)b
Chao1 Shannon InvSimpson Bray–Curtis

Resistome
ARG 0.093 0.015c 0.041c 0.022c
c c
ARG type 0.34 0.041 0.0022 0.057
MRG 0.94 0.31 0.59 0.076
ARG + MRG 0.13 0.041c 0.065 0.039c
Mobilome
MGE 0.15 0.18 0.041c 0.013c
Resistome + Mobilome
ARG + MRG 0.13 0.0087c 0.0022c 0.02c
+ MGE
ARG type + 0.59 0.0022c 0.0022c 0.018c
MRG + MGE
Mobile resistome
MGE + ARG 0.18 0.0022c 0.0022c 0.014c
type/MGE +
MRG
a
Mann–Whitney U test, Bonferroni–Holm p-adjustment method
b
PERMANOVA (10,000 permutations)
c
Significant difference between primary and secondary samples

(Gammaproteobacteria), but not Acidovorax (Betaproteo-

Fig. 6 Stacked barplots generated in R using relative abundances
computed according to Ma et al. (2016) compare (a) ARG composi-
tion at the ARG type level, (b) MRG composition and (c) MGE
composition between plasmid- and chromosome-derived reads of
primary and secondary wastewater. (PS (orange): plasmid secondary;
CS (orange): chromosome secondary; PP (blue): plasmid primary; CP
(blue): chromosome primary)
abundance of Acidovorax and Ruminococcus were not. Co-
incident rays of Prevotella and Bacteroides are interpreted as a
constant ratio of abundance. The unsupervised cluster den-
drogram and corresponding barplot (Fig. 4b) support the
observed grouping across wastewater treatment, but not several
projections from the compositional biplot. This is not surprising
given limits of the data projection, e.g., Aeromonas
bacteria), was a variable taxon, as clearly shown in Fig. 4b,
although Arcobacter (Epsilonproteobacteria), Bacteroides
(Bacteroidia), and Ruminococcus (Clostridia) were correctly
projected to be highly variable. Regardless of the discrepancies,
these interpretations support the conclusion that certain
microbial taxa distinguish primary from secondary wastewater.
Sixteen genera were statistically responsible for the
observed discrimination during wastewater treatment.
Seven genera representing major phyla except Actino-
bacteria were more abundant in primary wastewater. These
included environmental microbes such as Flavobacterium
and Oscillatoria (cyanobacteria), as well as bacteriophages
belonging to the family Myoviridae. Nine genera were more
abundant in secondary wastewater samples, including gut
(Prevotella, Bacteroides, and Ruminococcus) and environ-
mental (Acinetobacter, Sediminibacterium, Azonexaceae,
and Polaromonas) microbes. All major phyla were repre-
sented except Cyanobacteria and viruses, consistent with
the differences shown in Fig. 2a. Secondary wastewater
contained Nitrospira, Azonexaceae, and Tetrasphaera,
which are common in biological treatment of primary
wastewater (Cydzik-Kwiatkowska and Zielińska 2016;
Herbst et al. 2019). While the data will require actual via-
bility testing, the detection of Acinetobacter and Bacter-
oides in secondary wastewater effluents is concerning
especially since species in this genera are known pathogens
1582
with some exhibiting carbapenem and phenicol resistance
(Higgins et al. 2018; Niestępski et al. 2019).
Effect of wastewater treatment on the resistome
and mobilome
Diversity and composition differences between primary and
secondary wastewater were observed relevant to the resis-
tome, with more classes of AMR genes and MRGs in pri-
mary than secondary samples. The overall decreasing trend
in diversity along the wastewater treatment process is con-
sistent with observations from similar studies (Ng et al.
2019; Ben et al. 2017). However, this does not necessarily
support a decreased probability of AMR dissemination as
certain resistance genes like the OXA β-lactamases were
significantly enriched in the plasmid reads of secondary
samples. The observed enrichment in plasmids is not sur-
prising given that these enzymes, some of which are also
carbapenemases, are mostly carried in plasmids (Antunes
et al. 2014; Antonelli et al. 2015).
A comparison with published research using the ONT
MinION reveals both similarities and differences. The work
by Che et al. (2019) examining wastewater samples from
Hong Kong’s Shatin Wastewater treatment plant found
similar classes of ARGs, with dominance of genes con-
ferring resistance to aminoglycosides, beta-lactams, MLS,
tetracyclines, sulfonamides, phenicols, quinolones and
multidrug. However, with the exception of multidrug, a
higher percentage of each of these classes of ARGs was
located on plasmids, rather than on the chromosome. This is
in contrast to our work, which shows a larger portion of
ARGs associated with chromosome-derived sequences.
What is consistent with the Che et al. (2019) study is the
finding that a higher proportion of ARGs are present on
plasmids in secondary wastewater samples as opposed to
primary samples.
Multidrug, beta-lactam and peptide AMRs were the
dominant classes of ARGs in the NanoARG pipeline, and
overall there are more classes of AMR detected in primary
samples as opposed to secondary samples. However, total
numbers of ARGs were slightly increased for the March
secondary samples, and in terms of relative abundance,
secondary samples for both March and April were enriched
in OXA β-lactamases. This was confirmed by both the
CosmosID and Epi2Me bioinformatics pipelines. For Cos-
mosID, OXA β-lactamases represented ~12% and ~4% of
AMR genes in March and April primary samples, respec-
tively, and ~40% and ~50% of AMR genes in March and
April secondary samples, respectively. Epi2me uses differ-
ent algorithms and databases and identified a greater num-
ber of genes, but the trend was still consistent with OXA
β-lactamases representing ~1.5% of AMR genes in both
March and April primary samples, and ~11% and ~22% of
C. Martin et al.
AMR genes in March and April secondary samples,
respectively. This increase in β-lactamases through the
wastewater treatment process is consistent with other stu-
dies that highlight the risks of release of these ARGs to
receiving waters (Amador et al. 2015; Makowska et al.
2020).
As with ARGs, a larger proportion of MRGs were asso-
ciated with chromosome-derived sequences. MRGs also appear
to be enriched in secondary samples on the chromosome, but
not on plasmids. Li et al. (2015) examined plasmid ARGs and
MRGs from two Hong Kong wastewater plants, and found
zinc and copper to be the dominant MRGs, followed by cobalt
and arsenic. These genes were also found to be dominant in our
study in addition to MRGs for molybdenum and nickel, which
may reflect different sources. Li et al. suggest that high copper
and arsenic, used in animal breeding, may be present due to
slaughterhouse wastewater in one of the two WWTPs they
sampled. Although Amherst, MA, can be considered semi-
rural, with a population of only ~40,000, it does include a
major research university.
Mobilome genes detected in this study were mostly
located in plasmid-derived sequences. Genetic elements
related to transposase were the most common MGEs in all
samples examined. Transposases are implicated in DNA
mobility within and potentially between genomes and are
therefore important in horizontal gene transfer and bacterial
adaptation to new environments (Vigil-Stenman et al. 2017;
Cuecas et al. 2017). The class 1 integron-integrase intl1
gene was detected exclusively in all primary wastewater
samples and despite the very low occurrence, the observa-
tion is still consistent with the suggestion to use this gene as
an anthropogenic pollution marker (Gillings et al. 2015).
The finding that the secondary effluent samples have higher
mobilome diversity and composition does not immediately
increase the potential of clinically relevant transfer risks to
receiving environments, as these will be related to asso-
ciations with AMR elements. Clearly significant numbers of
resistome genes are either in plasmid-derived sequences or
are associated with other mobile genetic elements, with
implications for mobilization of antimicrobial resistance
(Slizovskiy et al. 2020).
Several of the MGE- and plasmid-associated resistance
genes for peptide antibiotics, beta-lactams, multidrug,
aminoglycosides, sulfonamide and tetracyclines were mat-
ched by the NanoARG pipeline to sequence reads of well-
known human pathogens like Clostridium botulinum,
Klebsiella pneumoniae, Escherichia coli, Acinetobacter sp.,
Staphylococcus aureus, Aeromonas hydrophila, Pseudo-
monas aeruginosa, Vibrio cholerae, Clostridioides difficile,
and Salmonella enterica. Interestingly, a number of those
reads fall under the current US CDC list of urgent and
serious threats of antimicrobial resistance (CDC AR Threats
Report 2019). For example, both primary and secondary
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . .
samples were found to contain MGE or plasmid-associated
reads corresponding to carbapenem-resistant Acinetobacter
which is at the top of the CDC list of serious threats. Car-
bapenems belong to the beta-lactam category of antibiotics
and are normally used as last resort drugs (Gupta et al.
2017). The finding that these reads were mobile, and hence
potentially transferable makes it even more concerning.
Interestingly, mobile reads corresponding to drug-resistant
Clostridioides difficile, another serious threat, were only
detected in primary samples. On the other side, mobile
reads corresponding to yet another serious threat,
carbapenem-resistant Enterobacteriacea, were mainly
detected in secondary samples. A number of reads in both
samples were also found to match mobile mcr-1, mcr-2,
mcr-3, mcr-4, and mcr-5 genes associated with pathogenic
species from bacterial orders Aeromonadales, Enter-
obacterales and Pseudomonadales. The mcr gene variants
confer resistance to colistin, which is used for severe
infections related to multidrug resistance (Liu et al. 2016).
Sufficient data suggests that the effluent samples in this
study contain the necessary components for the potential
spread of a clinically relevant AMR—clinically relevant
resistomes with transferable traits carried by serious human
pathogens. Traditional microbiology methods like culture-
based experiments will be needed to verify actual pathogen
viability and other factors like environmental selective
pressure will most likely play an important role in AMR
spread (Hall et al. 2015). Nonetheless, information gathered
in this study can provide the baseline to further probe the
potential AMR dissemination mechanisms in wastewater
effluents.
The differences in ARG and MRG locations between the
small Amherst wastewater treatment plant and much larger
Hong Kong treatment plants (Che et al. 2019; Li et al. 2015)
are not surprising and underscore the importance of study-
ing a wide variety of systems of different sizes, geographic
locations and sources of sewerage. We are at an early stage
in these studies in terms of reliable and reproducible
methodologies, but the ONT MinION provides a highly
functional WGS platform to conduct metagenomics studies
at different locations. The increasingly large number of
bioinformatics platforms produce challenges in reproduci-
bility of data due to the use of different algorithms and
databases. For site to site comparisons, it remains important
to use the same software packages for analysis.
Conclusions
The Oxford Nanopore Technologies MinION, coupled with
a variety of cloud-based bioinformatics workflows, provides
a useful, rapid and portable tool for characterizing complex
microbial communities and their resistome and mobilome
1583
profiles. Once characterized, the microbial communities can
be assessed with respect to the influence of treatment on
microbial composition. It is concluded that wastewater
treatment processes can be evaluated using the handheld
MinION, with the potential for future modeling of semi-
rural WWTPs without access to more sophisticated tools.
This in turn may help in evaluation of risks for receiving
waters, the environment, and downstream users, relative to
specific pathogens and AMR using data from the methods
presented here.
Funding This research was funded in part by Uniting for Health
Innovation, an “independent, nonprofit organization that unites gov-
ernment, industry, and local communities in the Americas to advance
innovation in public health.”
Author contributions Conceptualization: CM and TF; methodology:
CM and TF; software: CM and RC; validation: CM, BS, AC, AA,
NAH, RC, SH, and TF; formal analysis: CM, AA, SH, and TF;
investigation: CM, BS, and AC; resources: TF; writing—original draft
preparation: CM; writing—review and editing: CM, BS, AC, AA,
NAH, RC, SH, and TF; data visualization: CM; supervision: CM and
TF; project administration: CM and TF; funding acquisition: TF.
Compliance with ethical standards
Conflict of interest RC is the original Founder and Chair of Cosmo-
sID; NAH was the Chief Science Officer at CosmosID.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Afgan E, Baker D, van den Beek M et al. (2016) The Galaxy platform
for accessible, reproducible and collaborative biomedical ana-
lyses: 2016 update. Nucleic Acids Res 44(W1):W3–W10. https://
doi.org/10.1093/nar/gkw343
Amador PP, Fernandes RM, Prudêncio MC, Barreto MP, Duarte IM
(2015) Antibiotic resistance in wastewater: occurrence and fate of
Enterobacteriaceae producers of class A and class C β-lactamases.
J Environ Sci Health A Tox Hazard Subst Environ Eng 50
(1):26–39. https://doi.org/10.1080/10934529.2015.964602
An XL, Chen QL, Zhu D, Zhu YG, Gillings MR, Su JQ (2018) Impact of
waste water treatment on the prevalence of integrons and the genetic
diversity of integron gene cassettes. Appl Environ Microbiol 84(9):
e02766-17. https://doi.org/10.1128/AEM.02766-17
Antonelli A, D’Andrea MM, Vaggelli G, Docquier JD, Rossolini GM
(2015) OXA-372, a novel carbapenem-hydrolysing class D
β-lactamase from a Citrobacter freundii isolated from a hospital
wastewater plant. J Antimicrob Chemother 70(10):2749–2756.
https://doi.org/10.1093/jac/dkv181
Antunes NT, Lamoureaux TL, Toth M, Stewart NK, Frase H, Vaku-
lenko SB, Class D (2014) β-lactamases: are they all carbapene-
mases? Antimicrob Agents Chemother 58(4):2119–2125. https://
doi.org/10.1128/AAC.02522-13
Arango-Argoty G, Garner E, Pruden A, Heath LS, Vikesland P, Zhang
L (2018) DeepARG: a deep learning approach for predicting
antibiotic resistance genes from metagenomic data. Microbiome 6
(1):23. https://doi.org/10.1186/s40168-018-0401-z
1584
Arango-Argoty GA, Dai D, Pruden A, Vikesland P, Heath LS, Zhang
L (2019) NanoARG: a web service for detecting and con-
textualizing antimicrobial resistance genes from nanopore-
derived metagenomes. Microbiome 7(1):88. https://doi.org/10.
1186/s40168-019-0703-9
Ashbolt NJ, Amézquita A, Backhaus T et al. (2013) Human Health
Risk Assessment (HHRA) for environmental development and
transfer of antibiotic resistance. Environ Health Perspect 121
(9):993–1001. https://doi.org/10.1289/ehp.1206316
Ben W, Wang J, Cao R, Yang M, Zhang Y, Qiang Z (2017) Dis-
tribution of antibiotic resistance in the effluents of ten municipal
wastewater treatment plants in China and the effect of treatment
processes. Chemosphere 172:392–398. https://doi.org/10.1016/j.
chemosphere.2017.01.041
Berendonk TU, Manaia CM, Merlin C et al. (2015) Tackling antibiotic
resistance: the environmental framework. Nat Rev Microbiol 13
(5):310–317. https://doi.org/10.1038/nrmicro3439
Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein
alignment using DIAMOND. Nat Methods 12(1):59–60. https://
doi.org/10.1038/nmeth.3176
Cantón R, González-Alba JM, Galán JC (2012) CTX-M enzymes:
origin and diffusion. Front Microbiol. 3:110. https://doi.org/10.
3389/fmicb.2012.00110
CDC (2019) Antibiotic Resistance Threats in the United States, 2019.
U.S. Department of Health and Human Services, CDC, Atlanta,
GA. https://doi.org/10.15620/cdc:82532
Chahal C, van den Akker B, Young F, Franco C, Blackbeard J, Monis
P (2016) Pathogen and particle associations in wastewater: sig-
nificance and implications for treatment and disinfection pro-
cesses. Adv Appl Microbiol 97:63–119. https://doi.org/10.1016/
bs.aambs.2016.08.001
Che Y, Xia Y, Liu L, Li AD, Yang Y, Zhang T (2019) Mobile anti-
biotic resistome in waste water treatment plants revealed by
Nanopore metagenomic sequencing. Microbiome 7(1):44. https://
doi.org/10.1186/s40168-019-0663-0
Cuecas A, Kanoksilapatham W, Gonzalez JM (2017) Evidence of
horizontal gene transfer by transposase gene analyses in Fervi-
dobacterium species. PLoS ONE 12(4):e0173961. https://doi.org/
10.1371/journal.pone.0173961
Cydzik-Kwiatkowska A, Zielińska M (2016) Bacterial communities in
full-scale wastewater treatment systems. World J Microbiol
Biotechnol 32(4):66. https://doi.org/10.1007/s11274-016-2012-9
Czekalski N, Berthold T, Caucci S, Egli A, Bürgmann H (2012)
Increased levels of multiresistant bacteria and resistance genes
after wastewater treatment and their dissemination into lake
Geneva, Switzerland. Front Microbiol 3:106. https://doi.org/10.
3389/fmicb.2012.00106
D’Costa VM, King CE, Kalan L et al. (2011) Antibiotic resistance is
ancient. Nature 477(7365):457–461. https://doi.org/10.1038/na
ture10388
De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C
(2018) NanoPack: visualizing and processing long-read sequen-
cing data. Bioinformatics 34(15):2666–2669. https://doi.org/10.
1093/bioinformatics/bty149
Fernandes AD, Reid JN, Macklaim JM, McMurrough TA, Edgell DR,
Gloor GB (2014) Unifying the analysis of high-throughput sequen-
cing datasets: characterizing RNA-seq, 16S rRNA gene sequencing
and selective growth experiments by compositional data analysis.
Microbiome 2:15. https://doi.org/10.1186/2049-2618-2-15
Fouz N, Pangesti KNA, Yasir M et al (2020) The contribution of waste
water to the transmission of antimicrobial resistance in the
environment: implications of mass gathering settings. Trop Med
Infect Dis 5(1):33. https://doi.org/10.3390/tropicalmed5010033
Friedrich MJ (2016) WHO survey reveals misconceptions about
antibiotic resistance. JAMA 315(3):242. https://doi.org/10.1001/
jama.2015.18462
C. Martin et al.
Gillings MR, Gaze WH, Pruden A, Smalla K, Tiedje JM, Zhu YG
(2015) Using the class 1 integron-integrase gene as a proxy for
anthropogenic pollution. ISME J 9(6):1269–1279. https://doi.org/
10.1038/ismej.2014.226
Gloor GB, Reid G (2016) Compositional analysis: a valid approach to
analyze microbiome high-throughput sequencing data. Can J
Microbiol 62:692–703. https://doi.org/10.1139/cjm-2015-0821
Gloor GB, Macklaim JM, Pawlowsky-Glahn V, Egozcue JJ (2017)
Microbiome datasets are compositional: and this is not optional.
Front Microbiol. 8:2224. https://doi.org/10.3389/fmicb.2017.
02224
Gupta K, Gupta A, Shrivastava D (2017) The last resort antibiotics:
carbapenems. Int J Adv Res 5(4):1410–1413. https://doi.org/10.
21474/IJAR01/3957
Hall AR, Angst DC, Schiessl KT, Ackermann M (2015) Costs of
antibiotic resistance—separating trait effects and selective effects.
Evol Appl 8(3):261–272. https://doi.org/10.1111/eva.12187
Harnisz M, Korzeniewska E (2018) The prevalence of multidrug-
resistant Aeromonas spp. in the municipal wastewater system and
their dissemination in the environment. Sci Total Environ
626:377–383. https://doi.org/10.1016/j.scitotenv.2018.01.100
Herbst FA, Dueholm MS, Wimmer R, Nielsen PH (2019) The pro-
teome of Tetrasphaera elongata is adapted to changing condi-
tions in wastewater treatment plants. Proteomes 7(2):16. https://
doi.org/10.3390/proteomes7020016
Higgins PG, Hrenovic J, Seifert H, Dekic S (2018) Characterization of
Acinetobacter baumannii from water and sludge line of second-
ary wastewater treatment plant. Water Res 140:261–267. https://
doi.org/10.1016/j.watres.2018.04.057
Hultman J, Tamminen M, Pärnänen K, Cairns J, Karkman A, Virta M
(2018) Host range of antibiotic resistance genes in wastewater
treatment plant influent and effluent. FEMS Microbiol Ecol 94(4):
fiy038. https://doi.org/10.1093/femsec/fiy038
IACG (2018) Surveillance and monitoring for antimicrobial use and
resistance. Interagency Coordination Group (IACG) on Anti-
microbial Resistance discussion paper. 1–14.
Jain M, Olsen HE, Paten B, Akeson M (2016) The Oxford Nanopore
MinION: delivery of nanopore sequencing to the genomics
community [published correction appears in Genome Biol. 2016
Dec 13;17 (1):256]. Genome Biol 17(1):239. https://doi.org/10.
1186/s13059-016-1103-0
Kiełbasa SM, Wan R, Sato K, Horton P, Frith MC (2011) Adaptive
seeds tame genomic sequence comparison. Genome Res 21
(3):487–493. https://doi.org/10.1101/gr.113985.110
Kim D, Song L, Breitwieser FP, Salzberg SL (2016) Centrifuge: rapid
and sensitive classification of metagenomic sequences. Genome
Res 26(12):1721–1729. https://doi.org/10.1101/gr.210641.116
Kraemer SA, Ramachandran A, Perron GG (2019) Antibiotic pollution
in the environment: from microbial ecology to public policy.
Microorganisms 7(6):180. https://doi.org/10.3390/microorga
nisms7060180
Krawczyk PS, Lipinski L, Dziembowski A (2018) PlasFlow: pre-
dicting plasmid sequences in metagenomic data using genome
signatures. Nucleic Acids Res 46(6):e35. https://doi.org/10.1093/
nar/gkx1321
Lamba M, Ahammad SZ (2017) Sewage treatment effluents in Delhi: a
key contributor of β-lactam resistant bacteria and genes to the
environment. Chemosphere 188:249–256. https://doi.org/10.
1016/j.chemosphere.2017.08.133
LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ
(2011) Tertiary-treated municipal wastewater is a significant point
source of antibiotic resistance genes into Duluth-Superior Harbor.
Environ Sci Technol 45(22):9543–9549. https://doi.org/10.1021/
es202775r
Leonard AFC, Zhang L, Balfour AJ et al. (2018) Exposure to and
colonisation by antibiotic-resistant E. coli in UK coastal water
Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome. . .
users: environmental surveillance, exposure assessment, and
epidemiological study (Beach Bum Survey). Environ Int
114:326–333. https://doi.org/10.1016/j.envint.2017.11.003
Li H (2018) Minimap2: pairwise alignment for nucleotide sequences.
Bioinformatics 34(18):3094–3100. https://doi.org/10.1093/
bioinformatics/bty191
Li AD, Li LG, Zhang T (2015) Exploring antibiotic resistance genes
and metal resistance genes in plasmid metagenomes from was-
tewater treatment plants. Front Microbiol 6(Sep):1025. https://
doi.org/10.3389/fmicb.2015.01025
Liu YY, Wang Y, Walsh TR et al. (2016) Emergence of plasmid-
mediated colistin resistance mechanism MCR-1 in animals and
human beings in China: a microbiological and molecular biolo-
gical study. Lancet Infect Dis 16(2):161–168. https://doi.org/10.
1016/S1473-3099(15)00424-7
Luo G, Li B, Li LG, Zhang T, Angelidaki I (2017) Antibiotic resis-
tance genes and correlations with microbial community and metal
resistance genes in full-scale biogas reactors as revealed by
metagenomic analysis. Environ Sci Technol 51(7):4069–4080.
https://doi.org/10.1021/acs.est.6b05100
Ma L, Xia Y, Li B et al. (2016) Metagenomic assembly reveals hosts
of antibiotic resistance genes and the shared resistome in pig,
chicken, and human feces. Environ Sci Technol 50(1):420–427.
https://doi.org/10.1021/acs.est.5b03522
Makowska N, Philips A, Dabert M et al. (2020) Metagenomic analysis
of β-lactamase and carbapenemase genes in the wastewater
resistome. Water Res 170:115277. https://doi.org/10.1016/j.wa
tres.2019.115277
Mataseje LF, Boyd DA, Lefebvre B et al. (2014) Complete sequences
of a novel blaNDM-1-harbouring plasmid from Providencia
rettgeri and an FII-type plasmid from Klebsiella pneumoniae
identified in Canada. J Antimicrob Chemother 69(3):637–642.
https://doi.org/10.1093/jac/dkt445
McArthur AG, Waglechner N, Nizam F et al. (2013) The compre-
hensive antibiotic resistance database. Antimicrob Agents Che-
mother 57(7):3348–3357. https://doi.org/10.1128/AAC.00419-13
Mongan AE, Tuda JSB, Runtuwene LR (2020) Portable sequencer in
the fight against infectious disease. J Hum Genet 65:35–40.
https://doi.org/10.1038/s10038-019-0675-4
Newton RJ, McLellan SL, Dila DK, Vineis JH, Morrison HG, Eren
AM, Sogin ML (2015) Sewage reflects the microbiomes of
human populations. mBio 6(Feb):e02574–14. https://doi.org/10.
1128/mBio.02574-14
Ng C, Tan B, Jiang XT et al. (2019) Metagenomic and resistome
analysis of a full-scale municipal wastewater treatment plant in
Singapore containing membrane bioreactors. Front Microbiol
10:172. https://doi.org/10.3389/fmicb.2019.00172
Niestępski S, Harnisz M, Korzeniewska E et al. (2019) The emergence
of antimicrobial resistance in environmental strains of the Bac-
teroides fragilis group. Environ Int 124:408–419. https://doi.org/
10.1016/j.envint.2018.12.056
Numberger D, Ganzert L, Zoccarato L, Mühldorfer K, Sauer S,
Grossart HP, Greenwood AD (2019) Characterization of bacterial
communities in wastewater with enhanced taxonomic resolution
by full-length 16S rRNA sequencing. Sci Rep 9(Jul):9673.
https://doi.org/10.1038/s41598-019-46015-z
O’Neill J (2014) Antimicrobial resistance: tackling a crisis for the
health and wealth of nations. Rev Antimicrob Resist 20:1–16
Osińska A, Korzeniewska E, Harnisz M, Niestępski S (2017) The
prevalence and characterization of antibiotic-resistant and virulent
Escherichia coli strains in the municipal wastewater system and
View publication stats
1585
their environmental fate. Sci Total Environ 577:367–375. https://
doi.org/10.1016/j.scitotenv.2016.10.203
Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DG
(2014) BacMet: antibacterial biocide and metal resistance genes
database. Nucleic Acids Res. 42:D737–D743. https://doi.org/10.
1093/nar/gkt1252
Pehrsson EC, Tsukayama P, Patel S, Mejía-Bautista M, Sosa-Soto
G, Navarrete KM, Calderon M, Cabrera L, Hoyos-Arango W,
Bertoli MT, Berg DE, Gilman RH, Dantas G (2016) Inter-
connected microbiomes and resistomes in low-income human
habitats. Nature 533(May):212–216. https://doi.org/10.1038/
nature17672
Quach-Cu J, Herrera-Lynch B, Marciniak C, Adams S, Simmerman A,
Reinke RA (2018) The effect of primary, secondary, and tertiary
wastewater treatment processes on antibiotic resistance gene
(ARG) concentrations in solid and dissolved wastewater frac-
tions. Water 10(1):37. https://doi.org/10.3390/w10010037
Slizovskiy IB, Mukherjee K, Dean CJ, Boucher C, Noyes NR (2020)
Mobilization of antibiotic resistance: are current approaches for
colocalizing resistomes and mobilomes useful? Front Microbiol
11:1376. https://doi.org/10.3389/fmicb.2020.01376
Stokes HW, Gillings MR (2011) Gene flow, mobile genetic elements
and the recruitment of antibiotic resistance genes into Gram-
negative pathogens. FEMS Microbiol Rev 35(5):790–819. https://
doi.org/10.1111/j.1574-6976.2011.00273.x
Thi Khanh Nhu N, Riordan DW, Do Hoang Nhu T et al (2016) The
induction and identification of novel Colistin resistance mutations
in Acinetobacter baumannii and their implications. Sci Rep
6:28291. https://doi.org/10.1038/srep28291
Urban JM, Bliss J, Lawrence CE, Gerbi SA (2015) Sequencing ultra-
long DNA molecules with the Oxford Nanopore MinION.
bioRxiv. 019281. https://doi.org/10.1101/019281
Vigil-Stenman T, Ininbergs K, Bergman B, Ekman M (2017) High
abundance and expression of transposases in bacteria from the
Baltic Sea. ISME J 11(11):2611–2623. https://doi.org/10.1038/
ismej.2017.114
von Wintersdorff CJ, Penders J, van Niekerk JM et al. (2016) Dis-
semination of antimicrobial resistance in microbial ecosystems
through horizontal gene transfer. Front Microbiol 7:173. https://
doi.org/10.3389/fmicb.2016.00173
WHO (2014) Antimicrobial resistance: global report on surveillance.
World Health Organization. https://www.who.int/antimicrobial-
resistance/publications/surveillancereport/en/
Zhao S, Tyson GH, Chen Y et al (2015) Whole-genome sequencing
analysis accurately predicts antimicrobial resistance phenotypes
in Campylobacter spp. Appl Environ Microbiol 82(2):459–466.
https://doi.org/10.1128/AEM.02873-15
Wick RR, Judd LM, Holt KE (2018) Deepbinner: demultiplexing
barcoded Oxford Nanopore reads with deep convolutional neural
networks. PLoS Comput Biol 14(11):e1006583. https://doi.org/
10.1371/journal.pcbi.1006583
Xia Y, Li AD, Deng Y, Jiang XT, Li LG, Zhang T (2017) MinION
nanopore sequencing enables correlation between resistome
phenotype and genotype of coliform bacteria in municipal sew-
age. Front Microbiol. 8:2105. https://doi.org/10.3389/fmicb.
2017.02105
Zhang AN, Li LG, Ma L, Gillings MR, Tiedje JM, Zhang T (2018)
Conserved phylogenetic distribution and limited antibiotic resis-
tance of class 1 integrons revealed by assessing the bacterial
genome and plasmid collection. Microbiome 6(1):130. https://doi.
org/10.1186/s40168-018-0516-2